Dietary linoleic acid supplementation protects against obesity-induced microglial reactivity in mice

We investigated whether linoleic acid (LA) supplementation could modulate emotional behavior and microglia-related neuroinflammation. For that, male mice of C57BL/6J genetic background fed either a high-fat diet (HFD) or a standard diet (STD) for 12 weeks, were treated with a vehicle or LA solution for 5 weeks before being evaluated for emotional behavior using a battery of behavioral tests. The animals were subsequently sacrificed and their brains collected and processed for immunofluorescence staining, targeting microglia-specific calcium-binding proteins (IBA-1). Neuroinflammation severity was assessed in multiple hypothalamic, cortical and subcortical brain regions. We show an anxio-depressive-like effect of sustained HFD feeding that was neither alleviated nor worsened with LA supplementation. However, increased IBA-1 expression and microgliosis in the HFD group were largely attenuated by LA supplementation. These observations demonstrate that the anti-neuroinflammatory properties of LA are not restricted to hypothalamic areas but are also evident at the cortical and subcortical levels. This study discloses that neuroinflammation plays a role in the genesis of neuropsychiatric disorders in the context of obesity, and that LA supplementation is a useful dietary strategy to alleviate the impact of obesity-related neuroinflammation.

The multifactorial etiology of obesity obscures the underlying biological mechanisms 1 .Besides genetic vulnerability 2,3 , a large number of obesity cases in Western countries are the consequence of malnutrition, i.e., overconsumption of fat-rich food.Prolonged high-fat diets lead to the emergence of metabolic, cardiac, respiratory and endocrinal comorbidities 4,5 , and obesity has been correlated with neuropsychiatric disorders such as anxiety and depression 6,7 in clinical 8,9 and preclinical studies [10][11][12][13] .
Obesity has drawn much scientific attention since the crucial role of gut microbiota in human health was demonstrated 14 .Increased concentrations of short-chain fatty acids (SCAFs) released by the gut microbiota have been reported in individuals suffering from obesity 15 .Under physiological conditions, these products of colon microbial food fermentation induce the release of anorexigenic hormones into the peripheral circulation 16 , thus increasing circulating concentrations of leptin and insulin 17 .Since SCAFs can cross the intestinal epithelium to reach the portal vein and stimulate the vagal nerve, they may influence the homeostatic balance regulating individuals' appetite.Additionally, SCAFs modulate the immune response.They can reduce peripheral inflammation by inhibiting the production of pro-inflammatory cytokines 18 , and participate in the maturation of the microglia in the central nervous system (CNS) 19 .Subsequently, the abnormally elevated circulating SCAF concentrations in obesity correlate with higher levels of lipopolysaccharides (LPS).Elevated circulating SCAF concentrations and increased levels of LPS therefore correlate with the severity of the resulting systemic inflammation 20 .Additionally, the expression of gustatory receptors, which are sensitive to circulating LPS levels, is downregulated
We secondly studied the effect of 5 weeks of LA supplementation, starting from the 12th week of the differential regimen, on the animals' behavior.Contrary to expectations, no differences between the experimental groups were found, neither for HFD mice (HFD-LA: ST, grooming time:  2a-h).

Microglia reactivity evaluation through IBA-1 expression
Microglial reactivity was assessed through ionized calcium-binding adapter molecule 1 (IBA-1) staining.IBA-1 specifically targets microglia in the brain and is known to be upregulated during the activation of these cells.Microglial reactivity was evaluated in a selection of 6 hypothalamic regions (in rostral-to-caudal order: paraventricular nucleus-PVN; peduncular part of the lateral hypothalamus-PLH; ventromedial hypothalamus-VMH; lateral hypothalamus-LH; subthalamic nucleus-STN; parasubthalamic nucleus-PSTN), 5 cortical areas (prelimbic-PL; infralimbic-IL; orbitofrontal-OFC; anterior cingulate-ACC; anterior insular-aIC) and 9 subcortical regions (dorsolateral-DLS and dorsomedial-DMS striatum; dorsal region of the bed nucleus of the stria terminalis-dBNST; central-CeA and basolateral-BLA amygdala; dorsal hippocampus-dHipp; lateral habenula-LHb; ventral posteromedial thalamic nucleus-VPM; parvicellular part of the ventral posterior The effect of the regimen and LA supplementation on emotional behavior was evaluated during the last week of the experiment through a battery of tests: the splash test (ST), the nestlet shredding test (NST), the novelty-supressed feeding (NSF) task, and the light-dark box (LDB) test.Animals were then sacrificed for IBA-1 immunofluorescence staining.Created with BioRender.com.(b) All animals progressively gained weight during the experiment, and mice fed a HFD weighed significantly more than their control counterparts (STD) from the sixth week of the differential regimen.However, 5 weeks of LA supplementation did not affect the body weight of animals in either the HFD or the STD group.(c) A general effect of the diet on the weekly food intake (Kcal) was evidenced, with HFD mice consuming significantly more calories than control animals.Besides, LA supplementation did not significantly impact food intake in either obese (HFD-LA vs. HFD-VEH) or nonobese animals (STD-LA vs. STD-VEH).(d) Sustained fat intake significantly influenced the weekly beverage intake in mice, with HFD individuals drinking a significantly lower volume than control animals.However, LA supplementation did not influence this behavior in either obese (HFD-LA vs. HFD-VEH) or non-obese animals (STD-LA vs. STD-VEH).Values plotted are mean ± SEM (n = 10 per group).*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.thalamic nucleus-VPPC).The Bregma coordinates, the microglial density values for each brain region evaluated and the statistical comparison between experimental conditions are detailed in Table 1.The IBA-1 relative fluorescence intensity values and the statistical comparisons between experimental conditions are presented in Table 2.
The neuroinflammatory effect of the HFD was first evaluated in the hypothalamic brain structures.In terms of cellular density, a main effect of HFD was evidenced in all the regions evaluated, thus confirming a general microgliosis at the local level (Fig. 3).In regard to IBA-1-related fluorescence intensity, a main effect of HFD was The behavioral assessment using the novelty-suppressed feeding (NSF) task revealed no significant differences in emotionality among the experimental groups; all animals exhibited similar latencies in approaching the available food.(d-g) The light-dark box (LDB) test revealed the emergence of an anxious-like phenotype consequent to a sustained HFD, with obese animals (HFD-VEH) spending less time in the lit compartment than their control counterparts (STD-VEH) (d).This emotional alteration was not reflected in the other measured parameters, such as the frequency of visiting the lit compartment (e), the time in the transition area (f), and the traveled distance (g).No behavioral effect of 5 weeks of LA supplementation was observed in the LDB test (d-g), irrespective of the dietary regimen.(h) Sustained HFD did not induce the emergence of repetitive, compulsive-like behavior in mice, as evidenced in the nestlet shredding test (NST), and no impact of LA supplementation was revealed, irrespective of the regimen.Values are mean ± SEM (n = 10 per group).*, p < 0.05; **, p < 0.01; ns, not significant.
evidenced in the PVN, the PLH and the PSTN, as well as a tendency to increase in the STN.However, no differential IBA-1 expression between experimental conditions was found in the VMH, nor in the LH (Supplementary Fig. S1).Subsequently, the effect of the regimen was evaluated in the cortical structures.Microglial density of HFD individuals was significantly increased in all the cortical regions evaluated (Fig. 4).The comparison between fluorescence intensities also showed a global significant increase in IBA-1 expression at the cortical level, with the only exception being the IL (Supplementary Fig. S2).Finally, a significant increase of microglial density   www.nature.com/scientificreports/consequence of the HFD was evidenced in all the subcortical brain regions studied, with the only exception of the LHb (Fig. 5).IBA-1 relative expression was globally significantly increased at the subcortical level, with the exception of the VPPC, where only a tendency was detected, and of the dHipp and VPM, where no main effect of the regimen was evidenced (Supplementary Fig. S3).
The evaluation of the anti-inflammatory properties of LA supplementation in obese mice revealed an overall decrease in microglial density and IBA-1 expression.Hypothalamic microglial density significantly decreased after LA supplementation in all the structures evaluated (Fig. 3).Compared to the HFD-VEH group, HFD-LA animals showed reduced hypothalamic IBA-1-related fluorescence in the PVN and PLH, and a tendency in the PSTN.However, no significant effect of LA supplementation on IBA-1-related fluorescence was observed in the STN (no main effect observed in the VMH and LH) (Supplementary Fig. S1).Five weeks of LA supplementation also reduced obese mice's microglial density and IBA-1 expression at the cortical level, with the only exceptions being the aIC (Fig. 4) and the IL (no main effect revealed, Supplementary Fig. S2), respectively.Concerning the subcortical level, a global beneficial effect of LA administration was observed on HFD-induced microgliosis, though only a tendency was detected in the BLA, and no significant effect in the DLS (no main effect in the LHb) (Fig. 5).A significant decrease of IBA-1-related fluorescence intensity was observed in the DLS, dBNST, BLA, LHb and VPPC, as well as a tendency in the DMS and CeA (no main effect in the dHipp and VPM) (Supplementary Fig. S3).
Concerning LA supplementation in non-obese animals, microglial density was found unaltered regardless of the region evaluated (Figs. 3, 4 and 5).Concerning IBA-1 expression, LA supplementation increased relative fluorescence intensity only in the CeA, PSTN and STN of STD-LA mice compared to control animals, and a tendency was observed in the aIC, LHb and BLA.No other effects were observed (Supplementary Figs.S1-S3).

Morphometric evaluation of microglial reactivity
Microglial reactivity was further investigated through the morphological adaptations induced by HFD and LA supplementation.Microglial complexity in terms of cell ramification was evaluated in three brain structures, i.e. the PVN, the ACC and the BLA, respectively representing the hypothalamic, cortical and subcortical anatomical groups.

Evaluation of microglial contribution to anxio-depressive-like behavior
Principal component analyses (PCAs) were used to explore the relationship between animals' emotional behavior (LDB test and ST parameters, due to reported behavioral alteration), and microglial density and IBA-1 relative expression in hypothalamic, cortical and subcortical regions separately.
Based on the PCA results, 4 principal components (PC1-4) explain more than 75% of the total variance of each data set's metrics.As illustrated in Fig. 7, the first two PCs (PC1 and PC2) explain 58.64% of the total variance for the hypothalamic regions (Fig. 7a), 57.64% for the cortical regions (Fig. 7d) and 59.46% for the subcortical regions (Fig. 7g).The loading values (i.e.correlation values from data vs. eigenvectors) of PC1-4 representing the majority of the variation of the data sets are plotted as heat-maps for better interpretation of the contribution of single variables within single PCs.As a general pattern, microglial density and IBA-1 relative expression strongly correlate with PC1 in all hypothalamic (Fig. 7b), cortical (Fig. 7e) and subcortical regions (Fig. 7h), with PC1 increasing as microglial density and IBA-1 relative expression increase.Concerning the behavioral parameters, they mostly correlate with PC2 in hypothalamic and cortical regions, with the exception being the ST latency.In the subcortical data set, only the time spent in the lit compartment during the LDB test correlates with PC2.According to the PCA results, the observed increase in IBA-1 expression consequence of feeding the animals a HFD predicts local microgliosis in terms of cellular density.However, as illustrated in the PCA biplots, behavioral scores in the ST and the LDB test are only modestly predicted by the severity of microgliosis, irrespective of the anatomical location (Fig. 7c, f, i).The PCA scores of samples at the right of the PCA biplots (Fig. 7c, f, i) illustrate the similarity of the experimental groups in terms of the parameters evaluated, and show two main clusters.These clusters suggest that microglial reactivity might differently account for emotional

Discussion
The mechanisms underlying chronic neuroinflammation as a consequence of high fat consumption remain under-studied.Furthermore, excessive dietary fat intake and obesity enhance an individual's vulnerability to emotional disturbances 6,[10][11][12] .Our study shows that high-saturated-fat-induced neuroinflammation extends beyond the hypothalamus, significantly impacting cortical and subcortical brain regions.Neuroinflammation was observed concomitant to an anxio-depressive-like symptomatology in obese mice.Our work also confirms the anti-neuroinflammatory properties of LA supplementation and demonstrates its therapeutic potential at the chronic stage of neuroinflammation in a murine model of diet-induced obesity.
In line with previous studies 11,[30][31][32] , we show that sustained consumption of a fatty diet leads to a significant increase of the body weight, which to a certain extent, is a consequence of an installed hyperphagia.Although LA did not affect diet-induced obesity, it might have impacted energy and glucose homeostasis and therefore overall animal's metabolic health affecting, directly or indirectly, neuroinflammation and behavior.Since we know that our high-fat regimen leads to insulin resistance and peripheral inflammation, as show in our previous publication 30 , it would have been informative to include some physiological measurements like post-fasting glycemia in order to evaluate the contribution of LA supplementation at peripheral level.As expected, drinking intake was significantly reduced in obese individuals compared to non-obese animals, and therefore mean LA intake during the 5 weeks of treatment differed between the STD-LA and HFD-LA experimental groups.Future investigation should consider the effect of HFD on drinking intake and perhaps adapt the LA administration protocol, in order to address its contribution to inter-group differences.
The emotional disturbances observed here consequent to sustained high saturated fat consumption agree with several reports (e.g., 10 ).Compared to control mice, the grooming and lit compartment exploration times (in the ST and LDB, respectively) are significantly reduced in obese mice.However, no altered emotional behavior was observed in either the NSF task or NST.Therefore, the latency to first approach food could not sufficiently illustrate the motivational imbalance characterizing obese mice, and the HFD regimen did not induce compulsive-like behaviors.
Although beneficial effects of dietary supplementation on emotional disturbances have been reported 13,33 , neither anti-depressive nor anxiolytic effects of 5 weeks of LA supplementation were observed in our investigation.Interestingly, Demers et al. 13 demonstrated that emotionality in HFD-induced obese mice was alleviated when the regimen was supplemented with polyunsaturated ω-3 fatty acids; we did not come to the same conclusion, though we used an equivalent treatment duration.This difference might be related to the use of a ω-6 fatty acid instead of a ω-3 fatty acid, but also to the administration protocol, which differed between studies (oral vs. Compared to animals fed a standard diet (STD-VEH), the density of IBA-1 positive (IBA-1+) cells was significantly increased in mice after a prolonged HFD (HFD-VEH) at subcortical level: dorsomedial striatum (DMS) (a), dorsolateral striatum (DLS) (b), dorsal part of the bed nucleus of the stria terminalis (dBNST) (c), dorsal hippocampus (dHipp) (d), basolateral (BLA) (f) and central (CeA) (g) amygdala, ventral posteromedial thalamic nucleus (VPM) (h), and parvicellular part of the ventral posterior thalamic nucleus (VPPC) (i).No effect of the dietary regimen on microglial density was observed in the lateral habenula (LHb) (e).At the subcortical level, 5 weeks of LA supplementation significantly reduced microglial-related neuroinflammation in various structures (a, c, d, g-i), and a tendency to decrease was also revealed (f).Illustrative photomicrographs of BLA IBA-1+ cells for the experimental conditions STD-VEH (f1), STD-LA (f2), HFD-VEH (f3) and HFD-LA (f4), taken with a ZEISS Axio Imager Z2 microscope, equipped with ApoTome.2 and a Camera ORCA-Flash4.OLT (Zeiss, Germany), and processed to get maximal orthogonal intensity projections.Values are mean ± SEM (n = 6-8 per group).*, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.www.nature.com/scientificreports/gavage administration).Future complementary metabolic analyses might help us understand why 5 weeks of LA supplementation were not sufficient to improve the emotionality of obese mice.Increasing the LA treatment duration in a complementary study might also benefit the understanding of the discrepancies observed between microglial-related measurements and animals' emotionality.It would also be interesting to explore whether the combination of different polyunsaturated fatty acid leads to beneficial synergetic effects on animals' behavior.Obesity-related neuroinflammation has been described in hypothalamic regions due to their key role in energy homeostasis and food intake regulation (for a review, see 34 ).Interestingly, our study demonstrates that the inflammatory profile of obesity extends to the entire CNS.Microgliosis as a consequence of high-fat regimens occurs notably early: feeding a HFD for 1-4 days is sufficient to induce changes in microglia 35,36 .Nevertheless, literature alludes to the chronicity of neuroinflammation as a key feature for the emergence of emotional disturbances.We therefore assessed the anti-neuroinflammatory properties of LA, as well as its impact on obesityinduced emotionality 28,37 , while supplementing a high-saturated-fat diet.Five weeks of LA administration rescued overall cerebral microgliosis (HFD-VEH vs. HFD-LA) and switch the activation state of the microglial population (i.e. from hyper-ramified back to physiological-like morphology).Experimental HFDs have been shown show that microglial density and IBA-1 relative expression strongly correlate with PC1 for the three data sets (b, e, h), and that behavioral scores mostly correlate with PC2 in hypothalamic (b) and cortical regions (e).Only the time spent in the lit compartment during the LDB test correlates with PC2 at subcortical level (h).Biplots for each data set show IBA-1 relative expression predicts microglial density, and that ST and LDB behavioral scores are slightly predicted by the magnitude of microgliosis (c, f, i).Biplots illustrate two clusters of individuals that assemble in terms of microglial-related neuroinflammation.Thus, the pathological experimental group (HFD-VEH) shapes one of the clusters (c, f, i, red dots), and the second cluster comprises non-obese or obese animals where neuroinflammation had been rescued (c, f, i; respectively: STD-VEH, grey dots; STD-LA, bleu dots; HFD-LA, green dots).Entries in the lit compartment -LIT entries; time spent in the lit compartment, -LIT time; time in the transition zone, -TZ time; latency to first grooming event, -ST latency; total time grooming, -ST total.
to significantly reduce cerebral LA content (for instance 13 ).While LA metabolites participate in the control of the peripheral inflammatory response, LA has been suggested to modulate microglial function by binding free fatty acid receptors 1 and 4 (FFAR1 and FFAR4), and fatty acid binding proteins (FABPs) on their surface 38 .Hence, exogenous LA supplementation might have reversed the effect of HFD on brain LA content, and therefore rescued this anti-inflammatory pathway.Concerning our results, since we showed that LA exerts overall anti-inflammatory effects in the brain, an improvement in animals' emotionality was expected.Unfortunately, the behavioral of obese mice in the ST and the LDB test did not benefit from LA supplementation.The lack of behavioral effect might be attributed to the duration of the treatment.
Another noteworthy observation is the increased IBA-1 expression in the STN, PSTN, and CeA of control mice after LA supplementation.These regions participate in the regulation of food intake, and the PSTN in particular is involved in refraining from feeding 39,40 .Thus, the isolated LA effect suggests that these regions might be especially sensitive to the treatment and therefore, to dietary variations of fat intake.In addition to an under-characterized effect of central inflammation in terms of regional specificity and time course, the neuralglial dynamics of HFD-induced hypothalamic inflammation are not yet clearly understood.We hypothesize that STN and PSTN act as highly sensitive sensors of fat variations within the hypothalamus, potentially through CD36 fat-receptor expression, and enhance the inflammatory response through different synergistic effects of neural-originated and glia-originated inflammation.In fact, hypothalamic and extra-hypothalamic brain regions express CD36, allowing long-chain fatty acids to cross the blood-brain barrier and influence neuronal activity 41,42 .Further investigation on the effects of LA supplementation on other glial populations, and in particular, on astrocytes, which along with microglia are major actors of the neuroinflammatory response, would certainly help understanding the underlying processes.
In our exploration of the relationship between emotionality and the severity of neuroinflammation, we confirmed that an increase of IBA-1 expression in our model of diet-induced obesity predicted microgliosis in terms of cellular density.Although the PCAs results show that microgliosis is a poor predictor of animals' behavior in the LDB and the ST, individuals' PC scores aggregated into two clusters that differ in terms of the severity of microgliosis.Additionally, while linear models could not explain the overall variation of the data sets, they better predicted microgliosis through behavioral scores in untreated obese animals (see Supplementary data).These observations therefore suggest that the identification of microglial heterogeneity and signature is crucial in order to study the relationship between neuroinflammation and emotional imbalance.
Combined, our findings support the use of dietary LA supplementation as a therapeutic tool for alleviating neuroinflammation associated with overconsumption of fatty food and obesity.Our results open new vistas in the management of obesity by the supplementation of linoleic acid, a long-chain fatty acid, in dietary interventions.

Animals
Forty 8-week-old male C57BL/6JRj mice (Janvier Labs, Saint-Berthevin, France) were used in this study.Animals were group-housed and maintained under a controlled environment (12-h light/dark cycle from 7 a.m.; temperature: 22 ± 2 °C; humidity: 55 ± 10%).They had ad libitum access to food (standard diet-STD, or high-fat diet-HFD; see "Dietary regimens") and drinking water, vehicle or LA supplementation (see "Treatment").All procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH), the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, and the European Union regulations on animal research (Directive 2010/63/EU).They were approved by the University of Franche-Comté Animal Care and Use Committee (CEBEA-58).

Dietary regimens
Animals were randomly divided into two groups of 20 individuals each and were fed either an STD (KlibaNafag 3430PMS10, Serlab, CH-4303 Kaiserau, Germany) or a HFD (A03 food powder, SAFE, Augy, France; cholesterol C8667, Sigma-Aldrich, France; palm oil, Vigean, France) ad libitum as previously described 30 for 17 weeks.See diet composition details in Supplementary Table S1.

Treatment
After 12 weeks, mice were randomly divided as follows: half the animals in each group were treated with vehicle (VEH; 0.3% w/v, Arabic gum solution, in water; G9752, Sigma-Aldrich, France; STD-VEH, n = 10; HFD-VEH, n = 10), and the remainder were treated with a LA solution (0.2% LA, w/v, in VEH; L1376, Sigma-Aldrich, France; STD-LA, n = 10; HFD-LA, n = 10).The Arabic gum, which is an edible mixture of polysaccharides and glycoproteins, was used here as suspending agent since LA is immiscible in water.The LA dose was chosen based on previous studies 30 .Solutions were prepared three times a week in order to minimize LA oxidation, and the beverage intake was regularly monitored.

Behavioral characterization
The behavioral tests started 17 weeks after the onset of the dietary regimens and after 5 weeks of VEH/LA administration.They were conducted during the light/inactive phase of the circadian cycle.

Nestlet shredding test (NST)
To evaluate repetitive, compulsive-like behavior, mice were tested in the NST, which can robustly measure the effects of different treatments on their natural and spontaneous nestlet shredding behaviour.Mice were individually placed into a cage with litter and a pre-weighed nestlet, and left undisturbed for 30 min.After the test completion, mice were replaced in their home cages and the remaining intact nestlet material was removed, www.nature.com/scientificreports/dried overnight and weighed.This measure was used to calculate the percentage of the total nestlet that had been shredded, which served as an index of compulsive-like behavior 43,44 .

Splash test (ST)
Depressive-like behavior was investigated by leveraging rodents' inherent drive to groom themselves during the ST.Briefly, after spraying the dorsal surface of its body with a 15% sucrose solution, each animal was individually placed for 5 min in an individual home cage with litter.A prolonged latency to starting the first grooming period and an increased total time of grooming were considered as indicative markers for a depressive-like phenotype, in accordance with the literature 45 .

Novelty-suppressed feeding (NSF) task
In rodents, the measure of hyponeophagia (i.e., the inhibition of feeding produced by a novel environment) in the NSF task informs about emotional alterations by appealing to the appetitive component of incentive motivation 46 .The NSF task has been proved sensitive to pharmacological treatments targeting anxio-depressive symptomatology 47,48 .Following a 12-h period of food deprivation, animals were individually placed in an opaque cylindrical open-field apparatus (height: 60 cm; diameter: 47 cm), in the center of which they could reach two grain-based pellets located on a white piece of filter paper (diameter: 10 cm).Prolonged latencies until their first approach towards the food served as a quantitative indicator of anxio-depressive-like behavior.The test lasted for a maximum of 10 min (adapted from 49 ).

Light-dark box (LDB) test
In the LDB test, anxious-like behavior was assessed by subjecting animals to a choice paradigm involving an internal conflict between remaining in a dimly lit "safe" compartment (width: 40 cm; high: 14 cm; 20-30 lx) and venturing into a brightly lit "novel" one (width: 40; high: 26; 120-130 lx).The test duration was set at 10 min, during which the animal's behavior was continuously monitored and recorded using an automatic videotracking system (Viewpoint Behavior Technology, Lyon, France).The behavioral metrics evaluated were the duration and frequency of entries into the lit compartment, the time spent in the transition zone, and the total distance traveled within the testing arena.An entry was defined as the placement of all four of the animal's legs into either compartment.Otherwise, the animal was considered to be in the transition zone (adapted from 31 ).A shorter exposure to the brightly lit compartment compared to the dimly lit compartment was considered as indicative of anxious-like behavior.

Data and statistical analyses
The results are presented as means ± SEM.The statistical analyses were conducted using STATISTICA 10 (Statsoft, Palo Alto, United States).Figures were designed using GraphPad Prism version 10.2.0 for Windows (GraphPad Software, Boston, Massachusetts USA, www.graph pad.com).Assumptions for parametric analysis were verified using Shapiro-Wilk and Levene's tests to respectively study the data sets' normality of distribution and homogeneity of variance.The progression of body weight, food intake and liquid intake was evaluated with a repeatedmeasures ANOVA (RMA) design, including two (STD and HFD) or four (STD-VEH, STD-LA, HFD-VEH, and HFD-LA) groups (between-subject factor: regimen or treatment) and 12 or five measurements (within-subject factor: time).When the data sets did not meet assumptions for parametric analysis (behavioral, cellular density, immunofluorescence scores and morphometric scores), Kruskal-Wallis (KW) and Mann-Whitney U (MWU) tests were used.Gehan-Breslow Wilcoxon (GBW) tests were used to compare NSF score curves.Morphometric analyses aiming at evaluating microglia's morphological complexity in terms of ramification (i.e.endpoints) and perimeter of action (i.e.process length) were performed using ImageJ Fiji software (National Institute of Health, Bethesda, Maryland, USA, https:// imagej.net/ ij/ 53 ) as reported by Young and Morrison 54 .Principal component analyses (PCA) were performed in order to reduce redundancy of the information within the data sets and to maximize explanatory variance across measured parameters.The PCAs aimed to identify relevant components in the relationship between behavioral and microglia-related scores.For all analyses, the threshold for statistical significance was set at p < 0.05 and dependencies with p ≤ 0.1 were considered as trends.

Figure 1 .
Figure 1.Experimental design and animal monitoring.(a) Animals were fed either a high-fat (HFD) or a standard (STD) diet for 12 weeks before being treated with vehicle (VEH) or a 0.2% linoleic acid (LA) solution for 5 weeks.The animal's body weight and consummatory behavior were monitored during the entire experiment.The effect of the regimen and LA supplementation on emotional behavior was evaluated during the last week of the experiment through a battery of tests: the splash test (ST), the nestlet shredding test (NST), the novelty-supressed feeding (NSF) task, and the light-dark box (LDB) test.Animals were then sacrificed for IBA-1 immunofluorescence staining.Created with BioRender.com.(b) All animals progressively gained weight during the experiment, and mice fed a HFD weighed significantly more than their control counterparts (STD)from the sixth week of the differential regimen.However, 5 weeks of LA supplementation did not affect the body weight of animals in either the HFD or the STD group.(c) A general effect of the diet on the weekly food intake (Kcal) was evidenced, with HFD mice consuming significantly more calories than control animals.Besides, LA supplementation did not significantly impact food intake in either obese (HFD-LA vs. HFD-VEH) or nonobese animals (STD-LA vs. STD-VEH).(d) Sustained fat intake significantly influenced the weekly beverage intake in mice, with HFD individuals drinking a significantly lower volume than control animals.However, LA supplementation did not influence this behavior in either obese (HFD-LA vs. HFD-VEH) or non-obese animals (STD-LA vs. STD-VEH).Values plotted are mean ± SEM (n = 10 per group).*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Figure 2 .
Figure 2. Effect of 5 weeks of linoleic acid (LA) supplementation on sustained high-fat diet (HFD)-induced emotional alterations.(a) Even if the latency of the first grooming event in the splash test (ST) was similar for both experimental groups, (b) a depressive-like phenotype consequent to a sustained HFD was evidenced by ST, with obese animals (HFD-VEH) spending less time grooming themselves than those fed a standard diet (STD-VEH).(a, b) Linoleic acid supplementation affected the behavior of neither obese mice (HFD-LA) nor nonobese animals (STD-LA) in the ST.(c) The behavioral assessment using the novelty-suppressed feeding (NSF) task revealed no significant differences in emotionality among the experimental groups; all animals exhibited similar latencies in approaching the available food.(d-g) The light-dark box (LDB) test revealed the emergence of an anxious-like phenotype consequent to a sustained HFD, with obese animals (HFD-VEH) spending less time in the lit compartment than their control counterparts (STD-VEH) (d).This emotional alteration was not reflected in the other measured parameters, such as the frequency of visiting the lit compartment (e), the time in the transition area (f), and the traveled distance (g).No behavioral effect of 5 weeks of LA supplementation was observed in the LDB test (d-g), irrespective of the dietary regimen.(h) Sustained HFD did not induce the emergence of repetitive, compulsive-like behavior in mice, as evidenced in the nestlet shredding test (NST), and no impact of LA supplementation was revealed, irrespective of the regimen.Values are mean ± SEM (n = 10 per group).*, p < 0.05; **, p < 0.01; ns, not significant.

Figure 7 .
Figure 7. Relationship between behavioral scores in the light-dark box (LDB) test and the splash test (ST), and microglial-related neuroinflammation in the studied brain regions.From the overall results, the principal component analyses (PCAs) revealed that four principal components (PC1-4) explain 75% of the variance of the data sets (hypothalamic, a; cortical, d; and subcortical regions, g).Heat-maps of the loading values of PC1-4show that microglial density and IBA-1 relative expression strongly correlate with PC1 for the three data sets (b, e, h), and that behavioral scores mostly correlate with PC2 in hypothalamic (b) and cortical regions (e).Only the time spent in the lit compartment during the LDB test correlates with PC2 at subcortical level (h).Biplots for each data set show IBA-1 relative expression predicts microglial density, and that ST and LDB behavioral scores are slightly predicted by the magnitude of microgliosis (c, f, i).Biplots illustrate two clusters of individuals that assemble in terms of microglial-related neuroinflammation.Thus, the pathological experimental group (HFD-VEH) shapes one of the clusters (c, f, i, red dots), and the second cluster comprises non-obese or obese animals where neuroinflammation had been rescued (c, f, i; respectively: STD-VEH, grey dots; STD-LA, bleu dots; HFD-LA, green dots).Entries in the lit compartment -LIT entries; time spent in the lit compartment, -LIT time; time in the transition zone, -TZ time; latency to first grooming event, -ST latency; total time grooming, -ST total.